Bacteria rapidly develop resistance to antibiotic drugs within years of their introduction to the clinic1. Antibiotic resistance can be acquired by horizontal gene transfer or can result from persistence, in which a small fraction of cells in a population exhibits a non-inherited tolerance to antimicrobials2. Since antimicrobial drug discovery is lagging behind the evolution of antibiotic resistance, there is a pressing need for new antibacterial therapies3.
Bacterial infections are responsible for significant morbidity and mortality in clinical settings3. Though the advent of antibiotics has reduced the impact of bacterial diseases on human health, the constant evolution of antibiotic resistance poses a serious challenge to the usefulness of currently available antibiotic drugs3-7. Infections that would have been easily cured by antibiotics in the past are now able to survive to a greater extent, resulting in sicker patients and longer hospitalizations5, 8, 9. The economic impact of antibiotic-resistant infections is estimated to be between US $5 billion and US $24 billion per year in the United States alone10. Resistance to antibiotic drugs develops and spreads rapidly, often within a few years of first clinical use1. However, the introduction of new agents to the market by pharmaceutical companies has not kept pace with the evolution of antibiotic resistance1,3.
Acquired antibiotic resistance results from mutations in antibacterial targets or from genes encoding conjugative proteins that pump antibiotics out of cells or inactivate antibiotics11. Horizontal gene transfer, which can occur via transformation, conjugative plasmids, or conjugative transposons, is a major mechanism for the spread of antibiotic resistance genes12, 13. For example, Staphylococcus aureus became quickly resistant to sulpha drugs in the 1940s, penicillin in the 1950s, and methicillin in the 1980s12. In 2002, staphylococci developed resistance to vancomycin (the only uniformly effective antibiotic against staphylococci) by receiving vancomycin-resistance genes via conjugation from co-infecting Enterococcus faecalis, which itself became completely resistant to vancomycin in nosocomial settings by 198812, 14. Some agents (e.g., ciprofloxacin) promote the horizontal dissemination of antibiotic resistance genes by mobilizing genetic elements15, 16. Streptococcus pneumoniae and Neisseria gonorrhoeae have also obtained resistance to antibiotics (Morens, et al., (2004) Nature 430: 242-249). Sub-inhibitory concentrations or incomplete treatment courses can present evolutionary pressures for the development of antibiotic resistance17. Use of antibiotics outside of clinical settings, for example in livestock for the agricultural industry, has contributed to the emergence of resistant organisms such as methicillin-resistant staphylococci and is unlikely to abate due to economic reasons and modern farming practices12, 18. Resistance genes that develop in non-clinical settings may be subsequently transmitted to bacterial populations which infect humans, worsening the antibiotic resistance problem12.
In addition to acquiring antibiotic-resistance genes, a small subpopulation of cells known as persisters can survive antibiotic treatment by entering a metabolically-dormant state2, 19, 20. Persister cells do not typically carry genetic mutations but rather exhibit phenotypic resistance to antibiotics21. In Escherichia coli, the fraction of a population which represents persister cells increases dramatically in late-exponential and stationary phases. Chromosomally-encoded toxins may be important contributors to the persister phenotype but the underlying mechanisms that control the stochastic persistence phenomena are not well understood22-25. Persisters constitute a reservoir of latent cells that can begin to regrow once antibiotic treatment ceases and may be responsible for the increased antibiotic tolerance observed in bacterial biofilms20. By surviving treatment, persisters may play an important role in the development of mutations or acquisition of genes that confer antibiotic resistance.
Several strategies have been proposed for controlling antibiotic resistant infections. New classes of antibiotics would improve the arsenal of drugs available to fight antibiotic-resistant bacteria but few are in pharmaceutical pipelines3, 26. Surveillance and containment measures have been instituted in government and hospitals so that problematic infections are rapidly detected and isolated but do not address the fundamental evolution of resistance12. Cycling antibiotics is one method of controlling resistant organisms but is costly and may not be efficacious27, 28. Reducing the over-prescribing of antibiotics has only moderately reduced antibiotic resistance29. Efforts have been also made to lessen the use of antibiotics in farming but some use is inevitable30.
Using bacteriophage to kill bacteria has been in practice since the early 20th century, particularly in Eastern Europe16, 17. Bacteriophage can be chosen to lyse and kill bacteria or can be modified to express lethal genes to cause cell death31-35. However, bacteriophage which are directly lethal to their bacterial hosts can also produce phage-resistant bacteria in short amounts of time6, 7, 31, 32, 36. In addition to the aforementioned approaches, novel methods for designing antimicrobial drugs are becoming more important to extending the lifespan of the antibiotic era37. Combination therapy with different antibiotics or antibiotics with phage may enhance bacterial cell killing and thus reduce the incidence of antibiotic resistance, and reduce persisters38-41. Unmodified filamentous bacteriophage have been shown to augment antibiotic efficacy42. Systems biology analysis can be employed to identify pathways to target and followed by synthetic biology to devise methods to attack those pathways38, 43, 44.
Bacterial biofilms are sources of contamination that are difficult to eliminate in a variety of industrial, environmental and clinical settings. Biofilms are polymer structures secreted by bacteria to protect bacteria from various environmental attacks, and thus result also in protection of the bacteria from disinfectants and antibiotics. Biofilms may be found on any environmental surface where sufficient moisture and nutrients are present. Bacterial biofilms are associated with many human and animal health and environmental problems. For instance, bacteria form biofilms on implanted medical devices, e.g., catheters, heart valves, joint replacements, and damaged tissue, such as the lungs of cystic fibrosis patients97. Bacteria in biofilms are highly resistant to antibiotics and host defenses and consequently are persistent sources of infection98.
Biofilms also contaminate surfaces such as water pipes and the like, and also render other industrial surfaces hard to disinfect97. For example, catheters, in particular central venous catheters (CVCs), are one of the most frequently used tools for the treatment of patients with chronic or critical illnesses and are inserted in more than 20 million hospital patients in the USA each year. Their use is often severely compromised as a result of bacterial biofilm infection which is associated with significant mortality and increased costs.
Catheters are associated with infection by many biofilm forming organisms such as Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis and Candida albicans which frequently result in generalized blood stream infection. Approximately 250,000 cases of CVC-associated bloodstream infections occur in the US each year with an associated mortality of 12%-25% and an estimated cost of treatment per episode of approximately $25,000. Treatment of CVC-associated infections with conventional antimicrobial agents alone is frequently unsuccessful due to the extremely high tolerance of biofilms to these agents. Once CVCs become infected the most effective treatment still involves removal of the catheter, where possible, and the treatment of any surrounding tissue or systemic infection using antimicrobial agents. This is a costly and risky procedure and re-infection can quickly occur upon replacement of the catheter.